Project summary: The long-term objectives of this proposal are to establish generic techniques for membrane protein production and purification in different hosts and in formats that scale to high-throughput environments. Specifically, the GFP-based expression/purification pipeline recently set up for E. coli will be further developed (including a new detergent solubilization screen and improved vectors), and will also be implemented for the yeasts Saccharomyces cerevisiae and Pichia pastoris. A selection of S. cerevisiae membrane proteins will be put through the GFP pipeline in both hosts, and promising targets will be delivered for crystallization trials. The response of E. coli cells to overexpression of a set of bacterial and eukaryotic membrane proteins will be characterized by proteomics techniques, and strain engineering will be carried out based on the results obtained. Finally, a large number of E. coli operons containing predicted membrane proteins will be cloned and both homo- and hetero-oligomeric complexes will be identified by blue-native/SDS-PAGE analysis. Well-expressing complexes will be put through the GFP-based pipeline, and promising candidates will be delivered to crystallization trials. Relevance: Proper functioning of the cell membrane in terms of the import and export of molecules, the sending and receiving of signals, and the interactions with neighboring cells are absolutely essential to the health of the organism. Most of these functions are carried out by proteins in the cell membrane, yet, such proteins are difficult to produce and study biochemically. The project aims to develop better techniques for producing membrane proteins on a large scale.